The mechanism by which ATP is released from the central nerve terminals remains enigmatic. At the peripheral neuroeffector junction, the release is clearly or quantal, but several other mechanisms of release have been proposed. In the central nervous system, ATP has been variously reported to be co-released with GABA, and not released with glutamate. An elementary event of synaptic transmission is associated with a spontaneous release of a single quantum. On the postsynaptic membrane spontaneous neurotransmitter release is manifested by occurrence of miniature excitatory postsynaptic currents (mEPSCs). Thus the most unambiguous way to demonstrate quantal release of ATP is to record the specific P2X-mediated mEPSCs in the postsynaptic cells. In this study, we demonstrate that pyramidal cortical neurones exhibit a population of mEPSCs that are insensitive to TTX and to blockade of glutamate and GABA receptors. These mEPSCs are inhibited by P2X receptor antagonists, suggesting that they are mediated by ATP. The P2X mEPSCs are not synchronised with those mediated by glutamate- or GABA-mediated mEPSCs implying the existence of distinct pool of ATP-storing vesicles.